Translocation of the classic protein kinase C isoforms in porcine oocytes: implications of protein kinase C involvement in the regulation of nuclear activity and cortical granule exocytosis

Protein kinase C (PKC) is a family of Ser/Thr protein kinases categorized into three subfamilies: classical, novel, and atypical. The subcellular localization of classical PKCalpha, -betaI, and -gamma in the process of porcine oocyte maturation, fertilization, and parthenogenetic activation and their involvement in cortical granule (CG) exocytosis were investigated. The results of Western blot showed that PKCalpha, -betaI, and -gamma were expressed in the oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. Confocal microscopy revealed that the three PKC isoforms were concentrated in the GV but evenly distributed in the cytoplasm of MII eggs. PKCalpha and -gamma were translocated to the plasma membrane soon after sperm penetration. cPKCs migrated into the pronucleus in fertilized eggs. Following treatment with a PKC activator, phorbol 12-myristate 13-acetate (PMA), CGs were released and PKCalpha and -gamma were translocated to the membrane. The CG exocytosis and PKC redistribution induced by PMA could be blocked by the PKC inhibitor staurosporine. Parthenogenetic stimulation with ionophore A23187 or electrical pulse also induced cPKC translocation and CG exocytosis. Eggs injected with PKCalpha isoform-specific antibody failed to undergo CG exocytosis after PMA treatment or fertilization. The results suggest that cPKCs, especially the alpha-isotype, regulate nuclear function and CG exocytosis in porcine eggs.     

gamma-Amino butyric-N-acid sensitivity of mouse and human oocytes

gamma-Aminobutyric acid (GABA)-sensitivity was studied in mouse and human oocytes using electrophysiological techniques. Isolated mouse oocytes at the germinal vesicle (GV) or metaphase II stage, and human oocytes at the GV stage or following resumption of meiosis in culture, were sensitive to GABA. The transmitter usually hyperpolarized the membrane, with a concomitant decrease followed by an increase in membrane conductance, at threshold concentrations as low as 10(-10) M. GABA response was not evoked in Cl-free medium. Bicuculline (10(-5)-10(-6) M) reversibly blocked GABA (10(-9)-10(-4) M) responses. In contrast mouse fertilized eggs were insensitive to GABA at concentrations of 10(-5) M or lower. A possible biological role of the neurotransmitter GABA is discussed.     

Viable rabbits derived from reconstructed oocytes by germinal vesicle transfer after intracytoplasmic sperm injection (ICSI)

Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.     

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

MICROINJECTION OF TOAD SPERM INTO OOCYTES UNDERGOING MATURATION DIVISION*

Spermatozoa of Bufo bufo japonicus were briefly treated with Triton X-100 to remove their plasma membrane, and were injected into oocytes at various stages of maturation division. All the sperm injected into mature coelomic eggs transformed into pronuclei and synthesized DNA, as a normally fertilizing sperm does. The sperm injected into oocytes at the germinal vesicle (GV) stage did not show any change as long as the GV remained intact. In the oocytes which were induced to mature by progesterone, the injected sperm displayed characteristic features in synchrony with those of the resident female nucleus. These included the formation of several sperm-derived chromosomes in association with multipolar spindles in the oocytes from the stage of the germinal vesicle breakdown to the first polar spindle; the appearance of swollen, vesicular nuclei without concomitant DNA synthesis in those at the stage of the first polar body emission; and the reappearance of the condensed chromosomes with giant spindles in those at the stage of the second meiotic metaphase. Pricking of these last oocytes induced the formation of several male pronuclei and DNA synthesis. These results prove that the injection of detergent-treated sperm employed here provides an excellent means of studying the cytoplasmic state that regulates nuclear behavior.     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.     

Effect of age, GV transfer and modified nucleocytoplasmic ratio on PKCα in mouse oocytes and early embryos

Protein kinase C (PKC) is a family of Ser/Thr protein kinases that can be activated by Ca2+, phospholipid and diacylglycerol. There is evidence that PKC plays key roles in the meiotic maturation and activation of mammalian oocytes. The present study aimed to monitor the effect of age, germinal vesicle (GV) transfer and modified nucleoplasmic ratio on the subcellular distribution profile of PKCα, an important isozyme of PKC, in mouse oocytes undergoing meiotic maturation and following egg activation. Germinal vesicle oocytes were collected from 6-8-week-old and 12-month-old mice. Germinal vesicle-reconstructed oocytes and GV oocytes with one-half or one-third of the original oocyte volume were created using micromanipulation and electrofusion. The subcellular localization of PKCα was detected by immunocytochemistry and laser confocal microscopy. Our study showed that PKCα had a similar location pattern in oocytes and early embryos from young and old mice. PKCα was localized evenly in ooplasm, with weak staining in GV at the GV stage, and present in the entire meiosis II (MII) spindle at the MII stage. In pronuclear and 2-cell embryos, PKCα was concentrated in the nucleus except for the nucleolus. After the GV oocytes were reconstructed, the resultant MII oocytes and embryos showed a similar distribution of PKCα between reconstructed and unreconstructed controls. After one-half or two-thirds of the cytoplasm was removed from the GV oocytes, PKCα still had a similar location pattern in MII oocytes and early embryos from the GV oocytes with modified nucleoplasmic ratio. Our study showed that age, GV transfer and modified nucleocytoplasmic ratio does not affect distribution of PKCα during mouse oocyte maturation, activation, and early embryonic mitosis.     

Chromatin, microtubules, and kinases activities during meiotic resumption in bitch oocytes

In contrast to the majority of mammals, canine oocytes are ovulated at immature germinal vesicle (GV) stage and complete meiotic maturation to metaphase II during 48-72 hr within the oviducts. This study aims to characterize meiotic maturation process in bitch oocytes, with both morphological and biochemical approaches. The follow-up of chromatin and microtubules during maturation was described, and MPF and MAP kinase activities were quantified at different stages of maturation. Since bitch oocyte cytoplasm is darkly pigmented, the first step was to setup an appropriate staining method for DNA. We thus compared the efficiency of two visualization techniques and demonstrated that propidium iodide coupled to confocal microscopy was a better method than Hoechst/fluorescence microscopy for nuclear stage observation (determination rates: 98.6 vs. 69.5%, respectively; P < 0.01, n = 1622 oocytes). Microtubule organization, evaluated by tubulin immunodetection, revealed subcortical and perinuclear alpha-tubulin and asters in GV oocytes and a clear network of microtubules in GVBD oocytes. In MI and MII oocytes, a symmetrical, barrel-shaped, and radially located spindle was observed. MPF and MAP kinase activities were assayed concomitantly using histone H1 and MBP as substrates. Kinase activities were detected at low levels in oocytes at GV and GVBD stages and were significantly higher at MI and MII stages. In conclusion, despite the particular pattern of meiotic resumption in canine oocytes (ovulated at GV stage), cytoskeleton/chromatin organization and kinase activities follow a similar pattern to those observed in other mammalian species.     

The development of mouse oocyte cortical reaction competence is accompanied by major changes in cortical vesicles and not cortical granule depth

The basis for the incompetence of the cortical reaction in germinal vesicle stage (GV) mouse oocytes was studied by evaluating cortical granules (CGs) and vesicles in GV and mature oocyte cortices. Dark and light CGs had a similar mean distance of 0.4-0.6 micron from the plasma membrane for GV and mature cortices. The cortex of mature oocytes had a large population of membrane-bounded, 0.1-1.0 micron (diameter) vesicles. More than three times as many vesicles were observed in the CG domains of mature oocytes as were observed in GV oocytes. This lack of cortical vesicles (with their potential to store calcium) and not CG depth may account for cortical reaction incompetence in GV oocytes.     

Mouse-rabbit germinal vesicle transfer reveals that factors regulating oocyte meiotic progression are not species-specific in mammals

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0-14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage.     

Characterization of Polo-like kinase-1 in rat oocytes and early embryos implies its functional roles in the regulation of meiotic maturation,fertilization, and cleavage

Polo-like kinase 1 (Plk1) is a family of serine/threonine protein kinases that play important regulatory roles during mitotic cell cycle progression. In this study, Plk1 expression, subcellular localization, and possible functions during rat oocyte meiotic maturation, fertilization, and embryonic cleavages were studied by using RT-PCR, Western blot, confocal microscopy, drug-treatments, and antibody microinjection. Both the mRNA and protein of this kinase were detected in rat maturing oocytes and developing embryos. Confocal microscopy revealed that Plk1 distributed abundantly in the nucleus at the germinal vesicle (GV) stage, was associated with spindle poles during the formation of M-phase spindle, and was translocated to the spindle mid-zone at anaphase. In fertilized eggs, Plk1 was strongly stained in the cytoplasm between the apposing male and female pronuclei, from where microtubules radiated. Throughout cytokinesis, Plk1 was localized to the division plane, both during oocyte meiosis and embryonic mitosis. The specific subcellular distribution of Plk1 was distorted after disrupting the M-phase spindle, while additional aggregation dots could be induced in the cytoplasm by taxol, suggesting its intimate association with active microtubule assembly. Plk1 antibody microinjection delayed the meiotic resumption and blocked the emission of polar bodies. In conclusion, Plk1 may be a multifunctional kinase that plays pivotal regulatory roles in microtubule assembly during rat oocyte meiotic maturation, fertilization, and early embryonic mitosis.     

Identification of a translocation deficiency in cortical granule secretion in preovulatory mouse oocytes.

Preovulatory, germinal vesicle (GV)-stage mouse oocytes are unable to undergo normal cortical granule (CG) secretion. Full secretory competence is observed by metaphase II (MII) of meiosis and involves the development of calcium response mechanisms. To identify the deficient or inhibited step in CG secretion, preovulatory GV-stage oocytes were stimulated and tested for their ability to undergo translocation, docking, and/or fusion. The mean CG distance to the plasma membrane was not reduced in fertilized or sperm fraction-injected, GV-stage oocytes relative to that in control GV-stage oocytes. In addition, analysis of individual CG distances to the plasma membrane indicated no subpopulation of CGs competent to translocate. Further analysis demonstrated that secretory incompetence likely is not due to a lack of proximity of CGs to the egg's primary calcium store, the endoplasmic reticulum. Calcium/calmodulin-dependent protein kinase II (CaMKII), which is reportedly involved in secretory granule translocation and secretion in many cells, including eggs, was investigated. A 60-kDa CaMKII isoform detected by Western blot analysis increased 150% during oocyte maturation. The CaMKII activity assays indicated that MII-stage eggs correspondingly have 110% more maximal activity than GV-stage oocytes. These data demonstrate that the primary secretory deficiency is due to a failure of CG translocation, and that a maturation-associated increase in CaMKII correlates with the acquisition of secretory competence and the ability of the egg to undergo normal activation.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

Distribution of 5-chloromethylfluorescein diacetate staining during meiotic maturation and fertilization in vitro of mouse oocytes

The aim of this confocal microscopy study was to determine whether the pattern of CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) staining changes during meiotic maturation and fertilization in vitro of mouse oocytes. At different times during meiotic maturation and fertilization, oocytes, zygotes and two-cell embryos were stained with CMFDA to demonstrate intracellular glutathione S-transferase activity. After washing in CMFDA-free medium, most oocytes, zygotes and embryos were stained with dihydroethidium (HE) to visualize DNA structures. Meiotic maturation and fertilization in vitro of mouse oocytes were associated with changes in the pattern of intracellular CMFDA staining. In particular, accumulations of CMFDA-positive membranes were observed around the nucleus of germinal vesicle (GV) oocytes, overlaying the sperm nucleus as well as overlaying the first mitotic spindle if this approached the plasma membrane. Staining of oocytes and zygotes with the probes 3,3'-dihexyloxacarbocyanine iodine [DiOC6(3)], which stains all the intracellular membranes, and rhodamine 123, which stains active mitochondria, demonstrated that the intracellular structures evidenced by CMFDA staining did not correspond to accumulations of mitochondria. Exposure of oocytes and zygotes to the microtubule-disrupting agent nocodazole or the actin-depolymerizing drug cytochalasin D revealed an autonomous microfilament-dependent transport and relocation of CMFDA-positive membranes during meiotic maturation and fertilization. Such a transport of CMFDA-positive membranes may be envisaged as a protective shield built to prevent damage to DNA from endogenous and exogenous mutagen metabolites.     

Distribution and expression of phosphorylated histone H3 during porcine oocyte maturation

Phosphorylation modification of core histones is correlated well with diverse chromatin-based cell activities. However, its distribution pattern and primary roles during mammalian oocyte meiosis are still in dispute. In this study, by performing immunofluorescence and Western blotting, spatial distribution and temporal expression of phosphorylated serine 10 or 28 on histone H3 during porcine oocyte meiotic maturation were examined and distinct subcellular distribution patterns between them were presented. Low expression of phosphorylated H3/ser10 was detected in germinal vesicle. Importantly, following gradual dephosphorylation from germinal vesicle (GV) to late germinal vesicle (L-GV) stage, a transient phosphorylation at the periphery of condensed chromatin was re-established at early germinal vesicle breakdown (E-GVBD) stage, and then the dramatically increased signals covered whole chromosomes from pre-metaphase I (Pre-MI) to metaphase II (MII). Similarly, hypophosphorylation of serine 28 on histone H3 was also monitored from GV to E-GVBD, indicating dephosphorylation of histone H3 maybe involved in the regulation of meiotic resumption. Moreover, the rim staining on the chromosomes and high levels of H3/ser28 phosphorylation were observed in Pre-MI, MI, and MII stage oocytes. Based on above results, such stage-dependent dynamics of phosphorylation of H3/ser 10 and 28 may play specific roles during mammalian oocyte maturation.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

MEMBRANE REACTIONS DURING FERTILIZATION AND 1-METHYLADENINE STIMULATION IN IMMATURE STARFISH OOCYTES

Immature oocytes from starfishes were obtained during the breeding and non-breeding seasons. The oocytes were "fertilized" without previous induction of maturation by 1-methyladenine (1-MA) or germinal vesicle breakdown (GVB). Elevation of the fertilization membrane (FM) was observed with eggs obtained during the breeding season, but not observed with eggs obtained during the non-breeding season. Abnormal development was observed when these "fertilized" eggs with a FM were subsequently treated with 1-MA. The results of these experiments indicated that the elevation of FM was independent of GVB or the mixing of nuclear and cytoplasmic components.     

Morphological differences in nuclear materials released from hamster sperm heads at an early stage of incorporation into immature oocytes,mature oocytes,or fertilized eggs

To elucidate the effects of ooplasmic factors on the early morphological changes in hamster sperm heads within the ooplasm, immature ovarian oocytes at the germinal vesicle stage (GV oocytes), ovulated fully mature oocytes, and fertilized eggs at anaphase II or the pronuclear stage (PN eggs) were examined in detail 15–30 min after insemination or reinsemination. Thin-sectioning studies demonstrated distinct materials released from the sperm nucleus over the entire postacrosomal nuclear surface immediately after disappearance of the sperm nuclear envelope. The release occurred in all of the oocytes and eggs prior to or even in the absence of subsequent chromatin decondensation. Depending upon the stage of the penetrated oocyte or egg, however, the materials varied in morphology: several hemispherical projections of amorphous material within mature oocytes; a number of electron-dense globules within GV oocytes and PN eggs; and both forms within eggs at anaphase II-telophase II. These observations and the fact that only the release of the amorphous material was accompanied by sperm chromatin decondensation indicate that this release was the initial process of chromatin decondensation, whereas the release of the globules resulted from a deficiency or lack of ooplasmic factors affecting the sperm nucleus. Restriction of the release in both forms of material to the late meiotic phase suggests changes in the factors associated with progression of meiosis. To approach an understanding of the mechanism of successful decondensation of sperm chromatin, the ooplasmic factors considered responsible for the stage-dependent release of nuclear materials are discussed. © 1996 Wiley-Liss, Inc.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.